CA2-DI-(T-BUTYL)-1,4-BENZOHYDROQUINONE - RELATIONSHIP TO CA2+ POOLS AND RELEVANCE IN PLATELET ACTIVATION( RELEASE FROM PLATELET INTRACELLULAR STORES BY THAPSIGARGIN AND 2,5)

The effects of the Ca2+-ATPase inhibitors thapsigargin (Tg) and 2,5-di -(t-butyl)-1,4-benzohydroquinone (tBuBHQ) were examined by using Ca2+- regulatory systems of platelet mixed membranes, saponin-permeabilized and intact platelets. Both agents inhibit Ca2+-ATPase activities of pl atelet mixed membranes, without any effect on the basal Mg2+-ATPase ac tivity. Tg is more effective (EC50 = 35 nM) than tBuBHQ (EC50 = 580 nM ). The effect of the two inhibitors on Ca-45(2+) release from saponin- permeabilized platelets has also been characterized. Ca-45(2+) uptake into non-mitochondrial intracellular stores occurs via an ATP-dependen t mechanism, and if added at equilibrium the second messenger Ins(1,4, 5)P3 releases 50% of the accumulated Ca-45(2+). Maximally effective co ncentrations of Tg (1 muM) and tBuBHQ (50 muM) release 77% and 68% of the accumulated Ca-45(2+). Addition of Ins(1,4,5)P3 together with eith er Tg or tBuBHQ resulted in a non-additive release which was the same as with either Tg or tBuBHQ alone, indicating that the Ins(1,4,5)P3-se nsitive Ca2+ pool was a subset of the pool that is sensitive to the Ca 2+-ATPase inhibitors. Release of Ca-45(2+) by either Tg or tBuBHQ was not affected by heparin, which totally blocked Ins(1,4,5)P3-induced Ca 2+ release, and Tg was found not to affect [P-32]Ins(1,4,5)P3 binding to its receptor on mixed membranes. Thus both Tg and tBuBHQ release Ca 2+ from a pool that totally overlaps the Ins(1,4,5)P3-sensitive pool w ithout affecting Ins(1,4,5)P3 function. In intact indomethacin-treated Fura 2-loaded platelets, Tg and tBuBHQ cause Ca2+ elevation, arising from release from intracellular stores and influx from the outside. Bo th Tg and tBuBHQ elevated Ca2+ to similar levels, which were less and slower than those observed with thrombin. Addition of thrombin to cell s already treated with Tg or tBuBHQ produced further elevation of Ca2, indicating agonist utilization of a Ca2+-ATPase inhibitor-insensitiv e pool. In aggregation experiments Tg and tBuBHQ showed different func tional effects. In indomethacin-treated cells Tg induces slow aggregat ion and secretion responses, whereas tBuBHQ only induces shape change. Both agents show synergistic secretory responses with the protein kin ase C activator dioctanoylglycerol (DiC8). Tg also showed greater abil ity than tBuBHQ to release [H-3]arachidonic acid (AA) from [H-3]AA-lab elled platelets. Additionally, in [P-32]P1-labelled platelets both Tg and tBuBHQ induced phosphorylation of myosin light chain, a 27 kDa pro tein and the 45 kDa protein pleckstrin, but Tg showed a greater abilit y than tBuBHQ to cause phosphorylation of pleckstrin. These studies in dicate that Tg and tBuBHQ are effective in releasing the Ins(1,4,5)P3- sensitive Ca2+ pool in platelets. The differences obtained in Tg- and tBuBHQ-induced functional responses may reflect additional effects of Tg on protein phosphorylation.